The protein aggregation phenomena are prevalent throughout the industrial bioprocess. Proteins are expensive to express, isolate and purify due to their complex physical-chemical characteristics. Aggregation is considered a primary mode of protein degradation, often leading to immunogenicity in patients and a loss of efficacy. That is why the detection and determination of protein aggregates is a key point in the biopharmaceutical industry, as well as, in scientific research. The formation of protein aggregates is critical in industrial applications, because they can highly affect the production of protein therapeutics (i.e., biologics or biosimilars) effectively lowering the production yields.
U.S. Pat. No. 8,268,628 by Pastrana et al., teaches a method for determining the mechanism of aggregation and the amount of aggregation in protein, peptide or peptoid formulation, in solution or lyophilized state without the use of probes or additives by performing a novel FT-IR and 2DCOS analysis. FT-IR spectroscopy allows for a high degree of flexibility and speed in the determination of protein aggregates, with limited manipulation and without the use of exogenous probes. According to the '628 method, a sample is heated and left to equilibrate followed by spectral acquisition at the desired temperature and by following the method described by the '628 patent the determination of protein, peptide and peptoid, stability, aggregation and viability can be performed. The method has been applied to the study of lipids, membrane proteins, hydrophilic proteins, peptides and peptoids as a single component or in binary or ternary mixtures with other peptides, or lipid mixtures. When studying two protein components in a mixture or complex, one of the components must be isotopically labeled to allow for the simultaneous detection of each component. The flexibility of sample preparation, its potential for automation and data analysis are key elements of value for pharmaceutical protein formulation as illustrated in FIG. 11.
More specifically, the method described in the '628 patent uses FT-IR spectroscopy combined with the two-dimensional correlation analysis (2DCOS) which allows for the determination of the presence of aggregates, the determination of the mechanism of aggregation, allowing for correction in the pipeline manufacturing process of the protein to once again generate viable protein. In addition, the thermal transition of the protein can also be determined and a 2DCOS plot generated to compare with the established viable protein, allowing for quality control, stability and viability of the desired protein product. The ease of sample preparation and data analysis allows for the automation of this method.
However, FT-IR accessories for thermal dependence studies where both reference and sample are under identical temperature conditions are not commercially available. Normally, a user would have to perform the analysis of the reference then the sample and at times also collect water vapor spectra and perform tedious data manipulation.
Thus, there is a long felt need in the art for an FT-IR accessory suitable for thermal dependence studies where both reference and sample are maintained under identical temperature conditions.